Yeast arrays, methods of making such arrays, and methods of analyzing such arrays

ABSTRACT

This patent describes a novel method of detecting genetic interactions in yeast. This method can also be used to screen for function of biological effectors on yeast. The method encompasses crossing yeast strains with genetic alterations to acquire double mutants. The phenotypes of these double mutants are then checked to detect genetic interactions between the double mutants. This method can be used to assign function to yeast genes and their viral, prokaryotic, and eukaryotic homologs, and aptamers. It can also be used to study yeast two hybrid interactions and to find genes that regulate certain yeast promoters.

FIELD OF THE INVENTION

[0001] The present invention relates generally to genomics andproteomics. More specifically, it relates to high density output arraysof multiple yeast strains, methods of making the high density outputarrays, and methods of using the high density output arrays forfunctional analysis of genetic and protein-protein interactions.

BACKGROUND OF THE INVENTION

[0002] One of the major goals of the emerging field of proteomics is theestablishment of relationships between protein function and particulardiseases. Proteomic technologies are used to try to identify importantgenes and their related proteins implicated in diseases and theirtreatments and to understand the role these genes and their relatedproteins play in the onset and progression of disease. A majorproteomics challenge is to determine the set of proteins expressed inthe cell and the interactions between such proteins, which in turndefine the functional pathways of the cell. If a given pathway is linkedto a disease, then the proteins within the pathway or a functionallyrelated pathway may represent drug targets for treatment of the disease.

[0003] Accordingly, there is a need in the art for functional proteomicstechnologies which provide valuable functional information about genesencoding proteins with previously unknown roles. Yeast based proteomicsrepresents one such technology. Functional genomics and proteomicsstrategies involving large-scale construction of defined mutants havecreated the potential for the systematic mapping of genetic interactionson a genome-wide scale. In addition to the recent sequencing of thehuman genome, the genomes of other, simpler, organisms have beencompletely sequenced, including that of the budding yeast Saccharomycescerevesiae. For S. cerevisiae, deletion mutations have been constructedfor all 6,200 suspected genes, identifying a set of approximately 1200essential yeast genes and approximately 5,000 nonessential genes,resulting in approximately 5000 viable haploid gene deletion mutants.With genome sequence in hand, the monumental challenge is to understandthe roles of the approximately 6,200 predicted yeast gene products. Thescope of the challenge is immense. Approximately one-third of allpredicted yeast genes are classified as coding for proteins of unknownfunction [Saccharomyces Genome Database(http://genome-www.stanford.edu/Saccharomyces/)]. Further, among a testpool of 558 homozygous deletion strains, over 60% had no observablegrowth defect after 60 generations. Simple extrapolation to more complexgenomes suggests that the absence of obvious functions for a largefraction of encoded proteins will quickly become an enormous problem inbiology.

[0004] There is a need, therefore, for proteomics technologies which canassess the previously unknown functions of proteins. The phenotypicanalysis of the set of viable deletion strains within certain species ofyeast represents a major challenge because the role of many genes willonly be manifest under very specialized growth conditions. To addressthis problem, the present invention provides a high throughput methodfor the construction of yeast double-mutants that enables the phenotypeassociated with a specific mutation to be examined systematically withinthe context of thousands of different gene deletion backgrounds. Acomprehensive application of this method will identify the precisegenetic conditions under which each yeast gene is critical for fitnessof the organism and may reveal a conserved network of geneticinteractions linking fundamental processes and pathways of eukaryoticcells. Because many non-essential yeast genes have mammalian homologuessystematic synthetic lethal analysis on yeast will provide crucialinsights into the gene function problem in all eukaryotes. Suchsynthetic lethal analysis can be performed on the yeast arrays of thepresent invention. The high density yeast output arrays and the methodsof analyzing such arrays of the present invention therefore fulfill aneed in the art by providing simple and efficient methods forlarge-scale, high throughput analysis of genetic and protein-proteininteractions.

SUMMARY OF THE INVENTION

[0005] The invention is directed to compositions and methods forperforming large-scale analysis of genetic and protein interactions.

[0006] In one embodiment of the invention a high-density output array ofmultiple resulting yeast strains is constructed. Each resulting yeaststrain in the output array contains at least one resulting geneticalteration different from the genetic alterations in the other resultingyeast strains in the output array.

[0007] The resulting yeast strains in the output array are matingproducts of at least two input arrays. At least one of the input arrayscomprises multiple starting strains of yeast, each carrying at least onegenetic alteration, with the genetic alteration being different in eachstarting yeast strain. The starting and resulting yeast strains areselected from any yeast strain that has two mating types and is capableof mating and meiotic and mitotic reproduction. Examples of species thathave such strains are Saccharomyces cerevesiae and Schizosaccharomycespombe.

[0008] The input and output arrays are arranged on plates, with betweenabout 96 and about 6144 yeast colonies on one plate, and much higherdensities, over 10-fold higher, can be achieved if individual coloniesare pooled. The resulting strains in the output array are doublemutants. Two different types of output arrays are created, one in whichthe phenotypes associated with mutations (genetic alterations) areexamined within a diploid cell formed by mating the strains on the inputarrays and another in which mutations are examined within the context ofa haploid cell following sporulation of the diploids. The two mutationscan involve a mutation of two different endogenous yeast genes. Theseyeast genes can be non-essential yeast genes. Interactions betweendeleted genes in the output array can be discerned when the combinationof genes leads to either a synthetic lethal double mutant or a syntheticsick double mutant, i.e. where the double mutant grows more slowly thaneither of the single mutants on the input arrays. The entire outputarrays can comprise between about 1,000 and about 25 million resultingstrains of yeast, or between about 1 million and about 25 millionresulting yeast strains.

[0009] The input array contains starting yeast strains with startinggenetic alterations in at least one starting yeast strain. The geneticalterations can be of any of the following type: (i) an alteration inthe DNA encoding the gene such as a deletion or mutation of anendogenous essential or non-essential yeast gene; (ii) trans-dominantgenetic agents such as genes coding for nucleic acid or peptideaptamers, dominant-negative proteins, antibodies, small molecules,natural products, ribozymes, enzymes, RNAi, and antisense RNA or DNA;(iii) protein and RNA expression vectors of a heterologous gene from aviral, prokaryotic, or eukaryotic genome, wherein the genes can eitherbe wild type, mutated or fragmented (e.g. coding for a protein domain);(iv) a protein-protein interaction detection system, includingexpression plasmids coding for a two-hybrid interaction and reporterthat registers the interaction, the Ras recruitment system, thesplit-ubiquitin system, and various other protein fragmentcomplementation systems (e.g. DHR); and (v) a reporter whose expressionreflects a change in cellular state such as the activation or theinhibition of a pathway(s). The genetic alterations can be integratedinto the yeast genome or propagated on autonomously replicatingplasmids.

[0010] The aptamer can be either a peptide aptamer or a nucleic acidaptamer. It can either inhibit or enhance expression of genes, proteininteractions, or the activity of a protein or any other cellularcomponent.

[0011] The heterologous gene can be a human gene that can be a singlenucleotide polymorphism of another human gene.

[0012] In another embodiment of the invention, a high-density outputarray of resulting multiple yeast strains, where each resulting yeaststrain carries at least one resulting genetic alteration, and theresulting genetic alterations are different in each yeast strain, isconstructed through the method disclosed below. Multiple starting yeaststrains are generated, each strain carrying a starting geneticalterations. Sets of two starting yeast strains, each of the two setscontaining a different starting genetic alteration; are then mated. Themated strains are then made to undergo sporulation, resulting in haploidspore progeny. A single mating type is then germinated and the haploidspore progeny is cultured using selective growth criteria. Multiplehaploid yeast strains which carry a resulting genetic alteration whichis a combination of at least two starting genetic alterations areselected for through this process. The genetically altered yeast strainsare then arrayed in a high-density format on an output array.

[0013] The strains, plates, and genetic alterations used in thisembodiment are similar to the previous embodiment. The output arraydescribed in this embodiment could also be used to perform syntheticlethal analysis as described in the previous embodiment.

[0014] Yet another embodiment of the invention is a method forconducting small molecule screening of yeast colonies using ahigh-density input array of multiple starting yeast strains. The methodis carried out by generating an input array containing multiple startingyeast strains as described above. Then exposing this array to at leastone biological effector, and detecting change in phenotype in thestarting strains in response to the effector. The effector can be asmall molecule or any other biological effector. The input array andgenetic alterations can be the same array described in previousembodiments.

[0015] Yet another embodiment of the invention is a method forconducting synthetic lethal analysis of yeast colonies by producing aninput array of starting strains, and crossing the starting strains inthat array with other starting strains or another input array. Then thediploid resultant strains are studied for changes in phenotype due tothe combination of different genetic or chemical alterations.

[0016] The input arrays are constructed as detailed in the embodimentabove. The genetic alterations can be the same as the ones in theembodiments above. Chemical alterations can include biological effectorssuch as the ones described in the previous embodiment.

[0017] Yet another embodiment of the invention is a method to performsynthetic lethal analysis of yeast colonies of multiple yeast strainsusing DNA bar coding. In this method, starting strains are constructedas described above, but not placed into arrays. Each of the geneticalterations has a distinct DNA tag associated with it. This tag can be a20 nucleotide long DNA sequence associated with a certain geneticalteration. These starting strains are mated with other starting strainsof a different mating type, which generates the first output strain set,and then stimulated to undergo sporulation, which allows for selectedgrowth of haploid spore progeny that possess of both genetic alterationsand generates the second output strain set. The resulting output strainsare then studied and isolated through their genetic tags, dispensingwith the need to array each strain. The genetic alterations used in thisembodiment can be the same alterations used in the previous embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates the series of replica pinning procedures inwhich mating and meiotic recombination are used to generate haploiddouble mutants.

[0019]FIG. 2A illustrates the array of bni1Δ double mutants resultingfrom the final pinning and the corresponding wild-type control.

[0020]FIG. 2B illustrates that both the bni1Δ bnr1Δ and bni1Δ cla4Δdouble-mutants were inviable and that the bni1Δ bud6Δ double-mutant wasassociated with a slower growth rate or “synthetic sick” phenotype,reflecting reduced fitness of the double-mutant relative to therespective single mutants.

[0021]FIG. 3 illustrates a genetic network constructed from theinteractions listed in Table 1. A genetic interaction networkrepresenting the synthetic lethal/sick interactions is determined by thesynthetic genetic analysis of the present invention. Genes arerepresented as nodes and interactions are represented as edges thatconnect the nodes, 292 interactions and 205 genes are shown. The genesare colored according to their YPD cellular roles, with the mostabundant cellular roles shown.

[0022]FIG. 4 illustrates two dimensional clustering analysis ofsynthetic lethal interactions. The set of synthetic lethal interactionsassociated with mutations in 8 query genes, BIM1, BNI1, ARC40, ARP2,NBP2, BBC1, RAD27 and SGS1, were plotted on the horizontal axis, withthe query gene cluster tree above. The 201 genes that showed syntheticlethal interactions with the query genes were plotted on the verticalaxis with the cluster tree on the leftmost side of the plot. Syntheticlethal and synthetic sick (slow growth) interactions are represented asshades of red. An expansion of the plot is shown to allow visualizationof specific genes.

DETAILED DESCRIPTION

[0023] The present invention is directed to high-density output arraysof multiple yeast strains, methods for constructing such arrays, andmethods of using such arrays to conduct large-scale high throughputanalysis of genetic and protein-protein interactions. The presentinvention provides a systematic and efficient method for constructingarrays of yeast strains carrying multiple genetic alterations. Thisinvention enables a large-scale analysis of genetic and protein-proteininteractions, provides a method for validating potential drug targetsand for generating whole cell screens for compounds that perturb thefunction of these targets.

[0024] More specifically, the present invention provides compositionsand methods that will enable the systematic and automated constructionof arrays of yeast strains containing multiple mutations in ahigh-throughput manner. For example, the present invention could be usedto generate a high density output array of approximately 25 milliondouble mutant yeast strains, through crosses of input arrays involvingapproximately 5,000 viable haploid deletion mutants. The resultingoutput arrays can be used for large-scale analysis of genetic andprotein interaction networks by identifying the complete set ofsynthetic lethal double mutant combinations for a model eukaryotic cell.

[0025] The invention is directed to a variety of high-density yeastarrays, methods of making such arrays, and methods of using such arrays.The arrays include both input and output arrays. Input arrays are thosearrays used to generate output arrays. Input arrays are arrays ofmultiple yeast strains, with each yeast strain containing at least onegenetic alteration. These input arrays are crossed with either otherstarting yeast strains that contain at least one genetic alteration, orother input arrays to produce output arrays. Output arrays are thereforegenerated by crossing starting yeast strains from input arrays. Thestarting yeast strains each have at least one genetic alteration.Crossing of the starting yeast strains containing at least one geneticalteration results in strains of the output arrays containing at leasttwo genetic alterations. These genetic alterations present in both theinput and output arrays can include, but are not limited to, members ofthe group consisting of an aptamer, a system for detectingprotein-protein interactions, including a yeast two-hybrid system,expression of a heterologous gene from a viral, prokaryotic, oreukaryotic genome with the heterologous gene either having or not havinga yeast homolog, transformation with a promoter operably linked to areference gene, wherein the reference gene can be a reporter gene,mutation or deletion of an endogenous essential or non-essential yeastgene, or the addition of other dominant agents which can perturb anycellular function, including genes coding for dominant negativeproteins, antibodies, small molecules, natural products, ribozymes,RNAi, and antisense RNA or DNA.

[0026] As used herein, an input array is a grouping of a multitude ofstarting yeast strains located together on a solid support. In oneembodiment, the solid support is a plate. In a preferred embodiment,there are between about 96 and 6144 colonies on one plate, preferablythe array would be constructed at the highest density physicallypossible, which is limited by cell size and the number of geneticallydifferent strains that can be pooled and manipulated as one element ofthe array. The starting strains in the input array are selected from anyyeast species that has two mating types and is capable of meiotic andmitotic reproduction. In preferred embodiments, the starting yeaststrains can be from either the Saccharomyces cerevesiae species or theSchizosaccharomyces pombe species.

[0027] As discussed above, the genome of S. cerevisiae has beencompletely sequenced, and approximately 6,200 genes have been located.The international S. cerevisiae deletion consortium has constructedmutants corresponding to all of the approximately 6,200 suspected geneswithin the S. cerevisiae genome, with each mutant being a strain ofyeast containing a deletion of a different endogenous yeast gene. Tetradanalysis has revealed that approximately 15% of these deletion mutationsdefine genes essential for the viability of haploid cells, also referredto as “essential genes”. Thus, the deletion consortium identifiedapproximately 1,200 essential genes and approximately 5,000 nonessentialgenes.

[0028] The set of approximately 1,200 essential genes does not definethe minimal set of genes required for life because many genes that areindividually dispensable are not simultaneously dispensable. Syntheticgenetic interactions are usually identified when a specific mutant isscreened for second-site mutations that either suppress or enhance theoriginal phenotype. In particular, two genes show a “syntheticallylethal” interaction if they are associated with viability as singlemutations but combine to cause a lethal double-mutant phenotype. Thisphenotype in which a particular gene is only required for cell viabilitywhen another gene is also deleted is called a “synthetic lethalitydefect”. Similarly, the deletion of some genes results in a more severegrowth defect only when combined with the deletion of another gene, aphenotype referred to herein as a “synthetic fitness defect”. Becauseboth synthetic lethal defects and synthetic fitness defects representgenetic interactions, the reference herein to synthetic lethalinteractions or relationships also applies to synthetic fitnessinteractions.

[0029] Many synthetic lethal relationships appear to be specific,occurring for genes acting on a single biochemical pathway. They alsooccur, however, for genes within two distinct pathways if one processfunctionally compensates for defects in the other or if the twoprocesses are functionally related. Genetic screens for synthetic lethalinteractions traditionally involve three labor-intensive steps: (1)mutagenesis of a strain that carries a mutation in a “query” gene ofinterest; (2) isolation of mutants whose growth is dependent uponexpression of the query gene; and (3) cloning of the syntheticallylethal gene by complementation with a plasmid-born genomic library.Synthetic lethal screens have been applied successfully to identifygenes involved in cell polarity, secretion, DNA repair, and numerousother processes. On average, three to four different interactions wereidentified per screen, but very few of the published screens approachsaturation.

[0030] With a collection of defined mutations, synthetic lethalinteractions can also be identified through systematic construction andanalysis of double-mutants. The identification of syntheticlethal/fitness double-mutant combinations often indicates significant invivo interactions between gene products and serves as a key startingpoint for targeted biochemical or cell biological experiments. Indeed, asynthetic lethal/fitness mutant combination is often observed for genesthat impinge on the same essential cellular function. As describedabove, because the genome of S. cerevesiae is sequenced and most of theessential and non-essential genes are determined, yeast arrays crossingstrains with deletions or mutations of non-essential genes can begenerated in order to study the effects of double mutations.Accordingly, S. cerevesiae is an organism which works well in thehigh-density output yeast arrays of the present invention.

[0031] Additionally, the same methods described herein for the S.cerevisiae genome wide deletion set can be applied to other yeasts onceanalogous deletion sets are constructed. In particular, the fissionyeast Schizosaccharomyces pombe has proven an invaluable complementaryorganism to budding yeast for cell and molecular genetic analysis. Thecompletion of the fission yeast genome presents an enormous opportunityfor comparative biology between the two distantly related yeasts. Withinthe next several years, a complete gene deletion set will certainly beconstructed by the S. pombe community, and would be easily amenable tothe systematic synthetic lethal analysis using the large-scale geneticand protein interaction analysis methods of the present invention. Anyother fungal species that has two mating types can also be used with thepresent invention.

[0032] As stated above, the input array of the present inventioncontains a multitude of starting yeast strains. The array could contain,for example, about 5000 different yeast strains, each of which containsa different gene deletion. Such an input array could be crossed with asecond input array, which also contains about 5000 different yeaststrains, each of which also contains a gene deletion. This type ofoutput array containing approximately 25 million (5,000×5,000) differentyeast strains is described in detail in Example 5. Or, in anotherembodiment, an input array of about 5000 different yeast strains couldbe crossed with only one starting strain. Examples of these types ofcrosses are described in detail in Example 6. In yet another embodiment,more specific input arrays can be crossed with specific deletionmutants, as illustrated in Examples 7-10. The possibilities of differentcrosses between a first input array and a second input array or betweena first input array and a starting strain are quite numerous, and willbe discussed in more detail below.

[0033] In one embodiment of the invention, a first input array containsyeast strains, which are modified so that they contain at least onenon-lethal genetic alteration. The first input array is then crossedwith either another yeast starting strain or another input array, whichalso contains at least one genetic alteration, to form an output arraycontaining double mutants. Double mutants as used herein means yeaststrains which contain two genetic alterations, derived from singlemutants each of which contains at least one genetic alteration.

[0034] An output array is the product of a cross between either twoinput arrays or an input array and a starting strain. The input arraysand/or starting strain to be crossed are of different mating type, toallow for selection of the genetic alterations formed after the cross.For high-throughput synthetic lethal analysis with the set of viableyeast gene deletion mutants, the present invention provides a series ofreplica pinning procedures in which mating and meiotic recombination areused to generate two output arrays, one composed of diploid cellscarrying genetic alterations derived from the input arrays and anotheroutput array containing haploid meiotic progeny carrying geneticalterations derived from the input arrays (FIG. 1). In this scheme, aquery mutation is first introduced into a haploid starting strain, ofone mating type, and then crossed to the array of gene deletion mutantsof the opposite mating type. A set of diploids that are heterozygous forboth mutations represents the first output array that can be analyzedfor a phenotype. Sporulation of the diploid cells leads to the formationof double-mutant meiotic progeny and the second output array. Thestarting strain carries a reporter that allows for selected germinationof spores, which ensures that conjugation of meiotic progeny does notcomplicate the final analysis. Both the query mutation and the genedeletion mutations can be linked to dominant selectable markers, whichenables selected growth of double-mutants specifically. The finalpinning results in an ordered array of double-mutant strains, whosegrowth rate is monitored by visual inspection or image analysis ofcolony size.

[0035] The output arrays are generated by performing the followingsteps. The first step is to generate multiple starting yeasts strains,with each of these starting yeast strains carrying a genetic alteration.These starting yeast strains are then grouped into either two inputarrays, or one input array and a particular starting strain. The inputarrays are then crossed (mated), and diploid strains result, forming thefirst output array. The mated diploid strains then undergo sporulation,resulting in haploid strains. A single mating type is germinated. Thehaploid spore progeny that result from this sporulation are then grownusing selective growth criteria. Multiple haploid yeast strains, whichgrow on the selective media, can then be selected for the presence ofgenetic alterations that were in the starting strains. These recoveredhaploid yeast strains can then be arrayed in a high-density formatforming the second output array.

[0036] output array can contain hundreds, thousands, or millions ofresulting yeast strains with genetic alterations. In one embodiment, theoutput array contains between about 1,000 and 25 million resulting yeaststrains, and more preferably between about one and about 25 millionresulting yeast strains. Because the output array can be produced insuch a high-density format, containing millions of yeast strains, theoutput array can be used to assign gene function to multiple genessimultaneously. The high-density output array also allows forlarge-scale analysis of genetic and protein interactions, by analyzingthe phenotypes of the resulting strains within the output array.

[0037] One type of analysis that can be performed on the high-densityoutput arrays is a synthetic lethal/synthetic fitness analysis. Such ananalysis is used to determine the presence or absence of syntheticlethality defects or synthetic fitness defects. Synthetic lethalitydefect and synthetic fitness defect are phenotypes wherein either celldeath or retarded cell growth occurs only when two different genes aredeleted at the same time. When a specific gene is required for cellviability under conditions when a different gene is deleted or mutated,this resulting phenotype is termed a “synthetic lethality defect”. Thisphenotype is so named because the two genes deleted togethersynthetically lead to cell death. Similarly, the deletion of some genesresults in a more severe growth defect only when combined with thedeletion of another gene, a phenotype referred to as a “syntheticfitness defect”. Experience suggests that a fraction of the genes ofunknown function in S. cerevisiae will not exhibit synthetic lethalinteractions with other single deletions. However, this defined subsetrepresents the next logical target for a subsequent round of syntheticlethal screens that would yield genes that are essential only when twoother genes are deleted. A slight modification of the selection schemewill allow the synthetic lethal screens to be reiterated. By definition,when taken to the limit, this approach will identify the function of allgenes encoded by the yeast genome.

[0038] The identification of synthetic lethal/fitness double-mutantcombinations often indicates significant functional interactions betweengene products or the pathways containing the gene products and serves asa key starting point for targeted biochemical or cell biologicalexperiments. Accordingly, when a first input array is crossed witheither a second input array or a starting yeast strain, the phenotypesof the crosses are studied to see if the resulting strain develops asynthetic fitness, or synthetic lethal phenotype, or any otherdiscernible phenotype. In particular, the methods used in this inventioninclude the use of groups of fungal strains that contain geneticalterations. These strains can be crossed in order to study thephenotype of a strain with at least two genetic alterations.

[0039] The genetic alterations can be of any of the following types: (i)an alteration in the DNA encoding the gene such as a deletion ormutation of an endogenous essential or non-essential yeast gene; (ii)introduction of trans-dominant genetic agents such as genes coding forpeptide or nucleic acid aptamers, dominant-negative proteins,antibodies, small molecules, natural products, nucleic acid aptamers,ribozymes, enzymes, RNAi, and antisense RNA or DNA; (iii) protein andRNA expression vectors of a heterologous gene from a viral, prokaryotic,or eukaryotic genome, wherein the genes can be either wild type,mutated, or fragmented (e.g. coding for a protein domain); (iv) aprotein-protein interaction detection system, including expressionplasmids coding for a two-hybrid interaction and reporter that registersthe interaction, the Ras recruitment system, the split-ubiquitin system,and various other protein fragment complementation systems (e.g. DHR);and (v) a reporter whose expression reflects a change in cellular statesuch as the activation or the inhibition of a pathway(s). The geneticalterations can be integrated into the yeast genome or propagated onautonomously replicating plasmids.

[0040] The input arrays and starting yeast strains which are crossed toproduce an output array can contain within them a variety of geneticalterations, which allow for analysis of a variety of differentmodifications. In one embodiment of the invention, the yeast startingstrains in the input arrays have a gene deletion introduced into them astheir genetic alteration. This gene deletion could be of an essential,or non-essential gene. Non-essential gene deletions are deletions ormutations of genes that do not produce a lethal phenotype. InSaccharomyces cerevesiae, there are approximately 5,000 non-essentialgenes which, when either deleted or mutated, do not produce a lethalphenotype. Combinations of non-essential genes deletions can producesynthetic lethal or synthetic fitness phenotypes that will reveal howthese genes interact, what their function is in yeast i, and what thefunction of their homologs are in humans or other organisms.Combinations of these genes could also lead to other discerniblephenotypes, which could suggest the function of the deleted genes.

[0041] Essential genes are genes that when deleted or mutated cause alethal phenotype. The function of essential genes can be studied bymodulating their expression using inducible promoters. Similarly,conditional mutations can be introduced into essential genes allowingthe mutant phenotype to be analyzed under defined conditions. Forexample, temperature sensitive mutations are viable at a permissivetemperature and inviable at a restrictive temperature.

[0042] In one embodiment of the invention, an input array with anon-essential or essential gene deletion can then be crossed with otherstarting strains that contain other gene deletions, or any other geneticalteration. Genetic alterations mentioned in regards to this inventionmay include: non-essential gene deletions; essential gene deletions;aptamers; exogenous genes, either wild type, mutated, or fragmented(e.g. coding for a protein domain); genes coding for ribozymes; enzymes;RNAi, and antisense RNA or DNA; systems for detecting protein-proteininteractions such as the yeast two-hybrid system; and reporters whoseexpression reflects changes in cellular state.

[0043] In another embodiment of the invention, yeast-starting strains inthe input arrays have aptamers either integrated into their genome orintroduced as expression plasmids as their genetic alteration. Aptamersare peptide or nucleic acids that are produced through at leastpartially randomized pools of nucleic acid or amino acid sequences, thatare selected for their ability to bind certain epitopes. Peptideaptamers are defined as affinity agents that consist of constrainedcombinatorial peptide libraries displayed on the surface of scaffoldproteins. Peptide aptamers are trans-dominant agents that interact withgene products.

[0044] Ordered arrays of yeast strains expressing peptide or nucleicacid aptamers can substitute for arrays of yeast deletion strains. Inthis embodiment, starting strains containing gene deletions are crossedto an array of strains expressing peptide or nucleic acid aptamers andhaploid meiotic progeny expressing the peptide aptamer and carrying thegene deletion can be selected. The resulting strains that showaptamer-dependent synthetic lethality identify aptamers that inhibit agene product whose activity is required for viability of the startinggene deletion strain. The array of strains expressing peptide aptamerscould also be used to identify dominant inhibitors. In this case, astrain carrying a query mutation would be crossed to an array of strainsexpressing peptide aptamers and the resultant diploid cells examineddirectly for a phenotype. Those that show aptamer-dependent syntheticlethality as diploids would identify an aptamer that inhibits a genewhich shows a genetic interaction with the heterozygous query mutation.

[0045] Aptamers can inhibit the function of gene products by any one of,but not limited to only, the following mechanisms: (i) modulating theaffinity of a protein-protein interaction; (ii) modulating theexpression of a protein on a transcriptional level; (iii) modulating theexpression of a protein on a post-transcriptional level; (iv) modulatingthe activity of a protein; and (v) modulating the location of a protein.The precise mechanism of action of peptide aptamers can be determined bybiochemical and genetic means to ascertain their specific function inthe context of their interaction with other genes, and gene products.Strains carrying characterized aptamers can then be crossed with otherstarting strains that contain peptide aptamers or any other geneticalteration as described above. The phenotypes of these crosses can thenbe studied to determine if the resulting strains in the output arraydevelop a synthetic fitness, or synthetic lethal phenotype, or any otherdiscernible phenotype.

[0046] In another embodiment of the invention, the starting strainswould carry a heterologous gene or gene combination with a readout ofgene product activity. For example, the starting strain may contain aheterologous gene encoding an enzyme for which there is a biochemicalassay for its activity. In another example, the starting strain maycarry a yeast two-hybrid protein-protein interaction system or someother protein-protein interaction detection system such as the Rasrecruitment system, the split-ubiquitin system and various other proteinfragment complementation systems (e.g. DHR), which can be crossed toyeast within the input array that contain other yeast two-hybridinteraction systems, or any other genetic alteration. The geneticalterations in the other starting strain could be any of the onesdefined above or any other genetic alterations. The phenotypes of thesecrosses can be studied to determine if any of the resulting strainswithin the output array perturb the two-hybrid interaction. For example,if an input starting strain carries a set of genes that allow for theformation and detection of a two-hybrid interaction and the strainswithin the input array carry yeast gene deletions, then the output arraywould allow for the identification of deletions that perturb thetwo-hybrid interaction; or if the strains within the input array carrypeptides aptamers, then this system can be used to identify dominantinhibitory peptide aptamers that perturb the two-hybrid interaction.

[0047] In yet another embodiment of the invention, the starting strainsin the input arrays can express a heterologous gene(s) from either aprokaryotic, viral, or eukaryotic genome. In a preferred embodiment,heterologous genes are from the human genome. Different alleles of thesegenes can be tested in yeast to see how they interact with mutated yeastgenes that are homologous to human genes. This method can be used toassign function to different exogenous genes. Single nucleotidepolymorphisms (SNPs) of human genes could also be characterized toidentify SNP-dependent interactions. The genes of any organism could besimilarly manipulated with this system. These heterologous genes canreplace their deleted yeast homolog, or they can be genes that are nothomologous to any yeast gene. These exogenous genes could be crossedwith strains that contain either any of the genetic alterationsdescribed above, or any other genetic alteration.

[0048] Many yeast genes are conserved from yeast to humans and thus itis possible to functionally replace a yeast gene with its human homolog.There are many different alleles of a given human gene and some of thesemay be associated with a diseased state. The replacement of a yeast geneby set of alleles of its human homologue, each differing by one or moreSNP (single nucleotide polymorphism), in the context of the describedgenetic arrays offers a means to assess the functional interactions of agiven allele within a model eukaryotic cell. For example, the analysisof different alleles of a human gene may reveal that one allele inparticular is associated with a greater number of synthetic lethalinteractions, which suggests it is compromised for function relative toother alleles and, therefore, may be associated with a diseased state.If more than one conserved human gene is implicated in a diseased state,then in theory all combinations of different alleles can be tested forfunction within the context of a genetic array.

[0049] Yeast genetic arrays also permit the functional analysis ofheterologous genes that do not have yeast counterparts. For example,consider a human gene, designated hXXX, whose product is involved inreorganization of the actin cytoskeleton and for which there is no yeastcounterpart. Even though yeast cells do not contain a homolog hXXX,yeast cells have a highly conserved actin cytoskeleton and thereforewill likely contain gene products, such as actin, that the hXXX geneproduct may interact with. Thus, expression of the human gene within thecontext of a yeast genetic array will likely result in syntheticlethal/fitness defects that link the function of hXXX to actinreorganization. The heterologous gene could be taken from any viral,prokaryotic, or other eukaryotic genome.

[0050] Starting strains carrying heterologous genes can be crossed withstarting strains containing another exogenous gene, or any other geneticalteration. The genetic alterations in the other starting strain couldbe any of the ones defined above or any other genetic alterations. Thephenotypes of these crosses can then be studied to determine if theresulting strains in the output array develop a synthetic fitness, orsynthetic lethal phenotype, or any other discernible phenotype.

[0051] In another embodiment of the invention, the starting strains inthe input array contain a promoter from either a prokaryotic, viral, oreukaryotic genome operably linked to a reference gene. Starting strainsthat carry a promoter operably linked to a reporter gene can be crossedwith starting strains containing another promoter and reporter gene, orany other genetic alteration. The genetic alterations in the otherstarting strain can be any of the ones defined above or any othergenetic alterations. The phenotypes of these crosses can then be studiedto determine if the resulting strains in the output array develop asynthetic fitness, or synthetic lethal phenotype, synthetic dosagelethality, expression of the reporter gene, or lack thereof, or anyother discernible phenotype.

[0052] The ability to control the expression levels of genes withregulated promoters is especially useful for synthetic dosage lethalityscreens. Synthetic dosage lethality is a specialized version of aclassical synthetic lethality screen. In synthetic dosage lethality, areference gene is overexpressed in set of mutant strains carryingpotential target mutations. This reference gene can be an exogenous genefrom a viral, prokaryotic, or eukaryotic genome. More specifically, thegene could be a human gene. Increasing the amount of the reference geneproduct may not produce a phenotype in a wild-type strain. However, alethal phenotype may result when overexpression of a gene product iscombined with decreased activity of another gene product that impingeson the same essential function. For example, synthetic dosage lethalityhas been used to identify genetic interactions between CTF3, whichencodes a centromere binding protein, and a set of conditionalkinetichore mutants. The synthetic dosage lethality gene could alsocontain single nucleotide polymorphisms.

[0053] Genes can be overexpressed by cloning the open reading frame(ORF) behind a strong promoter, such as the galactose-induced GAL1promoter. Input arrays containing starting strains with theapproximately 5,000 different yeast deletions can be crossed to astarting strain carrying a plasmid that contains a GAL1-regulatedreference gene. Haploid meiotic progeny carrying GAL1-regulatedreference gene and carrying the gene deletion can be selected. Theoutput array containing yeast deletions combined with the GAL1-regulatedreference gene can be pinned from glucose medium, where the referencegene is not expressed, to galactose medium, where it is overexpressed,to score for synthetic dosage lethality.

[0054] In another embodiment of the invention, the gene-encodingreporter, such as green fluorescence protein (GFP), may be placed on aplasmid under the control of a regulated promoter. One useful promoteris the pheromone-induced FUS1 promoter, FUS1pr, which is massivelyinduced by stimulation of the MAP-kinase pathway that mediates thepheromone response in yeast. A FUS1pr-GFP gene could be constructed andintroduced into the set of yeast deletion mutants for expressionanalysis. Mutations that lead to increased basal levels of FUS1pr-GFPexpression, i.e. levels above that displayed by wild-type cells, wouldidentify genes that encode potential negative regulators of thepheromone response signal transduction pathway. By contrast, mutationsthat lead to decreased basal levels of FUS1pr-GFP expression wouldidentify genes that positively activate the activity of pheromoneresponse pathway signaling molecules. Quantitative analysis of GFPexpression can be achieved from agar-arrayed yeast colonies using afluorimager as demonstrated by studies using the genome reporter matrix(GRM) constructed by Acacia Biosciences. In the GRM, plasmid-borne GFPwas placed under the control of approximately 6,000 different yeastpromoters and a high-density array of yeast colonies (wild-type cells)carrying the reporter constructs was monitored for genome-wide changesin gene expression in response to drug treatments.

[0055] In yet another embodiment of the invention, each of the yeastdeletion mutants is constructed such that it is tagged with two unique20 mer oligonucleotide sequences. These “bar codes” allow foridentification and analysis of specific deletion mutants within largepopulations. A microarray printed with probes for the bar codes thatcorrespond to the approximately 5,000 viable deletion mutants can beused to follow synthetic lethality of particular strains following batchmating and sporulation experiments. Microarray-based synthetic lethalanalysis with bar-coded mutants follows the same series of stepsoutlined in the pinning procedure for double-mutant construction.However, in this case, the yeast mutants are manipulated as a pooledpopulation of cells and the growth of the cells is monitored as an arrayof bar codes. Thus, if a bar code is included into the components of thegenetic array, manipulations of the cells can be carried out in batchformat for array analysis via the bar codes. Details of the bar-codemethodology are described in Example 12.

[0056] The major advantage of the microarray approach to syntheticlethal analysis stems from its experimental simplicity. In themicroarray approach, the approximately 5,000 viable deletion mutants aremanipulated as a single pooled population, which eliminates the need forhigh-density arrays of mutants and the volumes of media associated withmanipulations of these arrays. Thus, with access to bar-codedmicroarrays and the pooled mutants, individual labs should be able tocarry out synthetic lethal screens rapidly. Overall, the paralleldevelopment of both the systematic and microarray-based approaches tosynthetic lethal screening will allow for exploitation of the strengthsof each strategy.

[0057] Described above are a variety of methods and composition forcomprehensive double-mutant construction in yeast that relies upongenetic manipulation to introduce a marked mutation or plasmid into anordered array of yeast mutants. In another embodiment, mutations andplasmids can be introduced into an input array via standardtransformation procedures, e.g. lithium acetate or electroporation forthe transformation of yeast cells. In this case, the resultanttransformants would form the output array.

[0058] Defined genetic alterations can be combined using geneticmanipulations or strain transformation protocols. In one embodiment ofthe invention, large-scale double-mutant combinations are constructedusing a specialized starting strain and an automated pinning method formanipulation of high density input arrays of defined starting yeastmutants. The manipulations to be performed can be performed by a robot.Such robotic manipulations are described in detail in the examples,including Example 2. The development of robotic methods for manipulationof the budding yeast genome-wide deletion set will set the stage forexciting uses of the present invention. The high-density output arraysof multiple yeast strains of the present invention can be used in avariety of ways to analyze genetic interactions on a large-scale, highthroughput basis. A description of some of these uses follows.

[0059] Synthetic Lethal Analysis of High Density Output Arrays AssigningGene Function

[0060] Mutations within many different yeast genes will lead to multiplesynthetic lethal/fitness defects, generating a synthetic lethal profilefor a given mutant. Cluster analysis of a set of synthetic lethalprofiles should identify mutant alleles that result in similarcompromised states and therefore perturb similar functions within thecell (FIG. 3 and FIG. 4). Thus, large-scale synthetic lethal/fitnessanalysis with yeast genetic arrays will provide a method of determininggene function. Mutations in nonessential genes are most easily analyzed;however, conditional alleles of essential genes, i.e. those genesrequired for cell growth, e.g. temperature sensitive alleles or thoseplaced under the control of a regulated promoter (e.g. the Tet-regulatedpromoter), can be analyzed for synthetic effects following introductioninto a genetic array. In the case of temperature sensitive alleles, theessential genes would be tested for synthetic lethal/fitness defects ata temperature below the nonpermissive/lethal temperature. For anessential gene under the control of a regulated promoter, syntheticlethal/fitness defects would be tested at an intermediate geneexpression level, i.e. at a level that compromises fitness but does notprevent cell growth.

[0061] Synthetic Lethal Analysis of High Density Output Arrays for DrugDiscovery and Potential Cancer Therapeutics

[0062] The Seattle Project at the Fred Hutchison Cancer Research Centeris founded on the concept that synthetic lethal analysis in modelorganisms provides a means to identify potential drug targets for cancertherapy. In this view, a null mutation represents a model for an idealdrug because in many cases it should mimic the effect of a highlyspecific inhibitor of the gene product. The identification of mutationsthat lead to a phenotype resembling a desired therapeutic outcome shouldthus also identify promising drug targets. Because the genes thatcontrol fundamental biological functions are often conserved, yeast andother model eukaryotes with sophisticated genetic methodology providepowerful systems for the identification of drug targets.

[0063] The rational identification of drugs for the treatment ofparticular forms of cancer or other diseases will likely stem from aknowledge of the mutations harbored by particular types of abnormalcells. In the context of anticancer drug therapeutics, the syntheticlethal analysis of a conserved yeast gene whose human homolog is ofteninactivated in tumors would identify potential targets for drug-inducedinhibition. In this view, synthetic lethal analysis identifies geneswhose inactivation results in cell death only within the context ofanother specific mutation and thus provides a potential program forkilling cells within a diseased context.

[0064] Synthetic Lethal Analysis of High Density Output Arrays toGenerate Cocktail Therapies

[0065] If synthetic lethal/fitness analysis identifies novel genedeletion mutations that show a synthetic effect in combination with aknown drug or mutant allele of a known drug target, then the identifiedgenes may represent a new target for a drug that will enhance theeffectiveness of the known drug. For example, if we identify genedeletion mutants that are hypersensitive to the growth inhibitoryeffects associated with Cisplatin, a cancer therapeutic agent, theninhibitors of the identified genes would represent potential targets fordrugs that would be used in combination with Cisplatin to kill cancercells. In another example, if we identify gene deletion mutants thatwere hypersensitive to an antifungal drug, then the inhibitors of theidentified genes would represent potential targets for drugs that wouldused in combination with the antifungal drug to kill a fungal pathogen.This procedure could also be used with any genetic manipulation eitherdefined above or elsewhere.

[0066] Synthetic Lethal Analysis of High Density Input Arrays ScreenedAgainst Small Molecules to Analyze Small Molecule-Target Interaction

[0067] Comprehensive synthetic lethal analysis should provide us with akey for deciphering the interactions between small molecules andbiological targets in yeast. In this scheme, the approximately 5,000viable yeast disruption mutants are screened against small molecules ofinterest for synthetic drug sensitivity. If the sensitivity to aparticular molecule resulted from the inactivation of a specific targetthen the synthetic drug sensitivity should mirror the synthetic lethalprofile of the target gene. Thus, a comprehensive synthetic lethalprofile provides a key for linking small molecules to biological targetsin a whole cell growth assay.

[0068] The results of the comprehensive synthetic lethal analysis inyeast provide a whole cell screen for inhibitors of specific targetmolecules. For example, to search for inhibitors of a the PAK-likekinase Ste20p, we would simply screen for small molecules thatspecifically kill the panel of yeast mutants carrying deletion mutationsthat are synthetically lethal when combined with a ste20Δ mutation. Thespecificity of the screen will depend upon the specificity of thesynthetic lethal profile associated with the ste20Δ mutation. Given thateach mutation will be tested for synthetic lethality in approximately5,000 different contexts, it is anticipated that even genes within thesame pathway may show a distinct synthetic lethal profile.

[0069] Suppressor Analysis of a Conditional Lethal Situation

[0070] Genetic arrays can be used to identify mutations that function assuppressors of lethality. For example, consider a mutation in a genewhose product functions within a DNA damage check point signaltransduction pathway. The combination of a DNA damage check point mutantand a DNA damaging agent leads to lethality and we can use a geneticarray to screen for gene deletion mutations that suppress theconditional lethal situation. As another example, consider a gene thatleads to lethality when overexpressed; in this case, we can use agenetic array to screen for mutations that are either hypersensitive orresistant to overexpression of the detrimental gene.

[0071] Genetic Mapping and Backcrossing the Yeast Deletion Mutationsinto Another Genetic Background

[0072] Use of the synthetic genetic arrays of the present inventionshould also allow for backcrossing the entire set of deletion mutationsinto another genetic background to analyze traits specific to thatbackground. Examples of genetic backgrounds which can be analyzedinclude the Σ1278 background that is competent for filamentous growthand the SK1 background that is hyperactive for sporulation.

[0073] Because haploid yeast double-mutants within the output array areformed by meiotic recombination, the analysis of strains within theoutput array can be used to map gain of function phenotypes, even thosethat are multigenic traits. In this case, the gene(s) associated withthe gain of function phenotype would fail to form double mutantsefficiently with genetically linked gene deletions, resulting in aseries of output strains that fail to inherit the gain of functionphenotype.

[0074] High Density Peptide Aptamer Mammalian Cell Microarrays

[0075] The synthetic genetic array analysis of the present invention canbe extended from yeast cells to mammalian cells by using an array oftransfection constructs that lead to the expression of peptide ornucleic acid aptamers or other genetic alterations. In the case ofpeptide aptamer expression, the peptide aptamer expression plasmids arefirst suspended in gelatin solution and arrayed on glass slides using arobotic microarrayer. Mammalian cells are then cultured on the glassslides containing the peptide aptamer expression plasmids. Cells growingin the vicinity of the gelatin spots uptake the peptide aptamerexpression plasmids creating spots of localized transfection within alawn of nontransfected cells. Once the peptide aptamer expressionplasmids are incorporated into cells they can function as dominantagents, dominant agents being agents which perturb the function of thecell in any way.

[0076] In one example, an input starting mammalian cell line might carrya set of genes that allow for the formation and detection of atwo-hybrid interaction and the input array might carry a set of peptideaptamer expression plasmids. In this case, the output array wouldconsist of mammalian cells transfected with the aptamers that mayperturb the two-hybrid interaction. The cell microarrays can be designedsuch that the positions of individual aptamers in the yeast array arecross-correlated to the positions of the same aptamers in the cellmicroarray. This correlation will allow aptamers that have observablephenotypes in the yeast array, much as the disruption of a mammalianprotein interaction, to be directly assessed in the cell microarray.

[0077] As described above, there are numerous uses for the high densityoutput arrays of multiple yeast strains of the present invention,including but not limited to synthetic lethal analysis of high densityoutput arrays to assign gene function, synthetic lethal analysis of highdensity output arrays for drug discovery and potential cancertherapeutics, synthetic lethal analysis of high density output arrays togenerate cocktail therapies, synthetic lethal analysis of high densityinput arrays screened against small molecules to analyze smallmolecule-target interaction, and suppressed analysis of a conditionallethal silation.

[0078] This invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Creation of Double Mutant Output Array

[0079] An example of a starting strain that could be used in thisembodiment is the Saccharomyces cerevesiae strain termed Y2454. TheY2454 strain is characterized by being a MAT□ mating type with ura3,leu2, his3, and lys2 mutations, and a HIS3 gene linked to an MFA1promoter. The ura3, leu2, his3, and lys2 mutations require the strain tobe grown in supplemented media to survive. They also carry a can1 nullallele which confers canavinine resistance to the cells. A mutant gene,for example, one of the approximately 5,000 non-lethal mutations foundin Saccharomyces cerevesiae, is introduced into this strain. The deletedgene is being replaced by a NAT gene which confers noureseothricinresistance to these cells.

[0080] This strain can be crossed with a starting array of yeast strainsof the MATa mating type. The strains in this starting array containura3, leu2, his3, and met15 knockouts, so that they can only survive onsupplemented media. These cells can also contain a mutation of one ofthe approximately 5,000 non-lethal gene deletions known in Saccharomycescerevesiae. The deleted gene is replaced with an operably linked KANgene, which gives the yeast cells resistance to kanamycin derivativeslike Geneticin. These starting array strains carry a wild-type CAN1locus, which makes them sensitive to canavinine.

[0081] A double mutant haploid cell could then be developed by thefollowing steps:

[0082] Step 1. Construction of a Y2454-derivative that carries anat-marked mutant allele, e.g. bni1Δ::nat where the given gene isdeleted and replaced with nat, which results innourseothricin-resistance, for genome-wide synthetic lethal analysis.

[0083] Step 2. Mating of the MATα Y2454-derivative to the array of MATaxxxΔ::kan deletion mutants on rich medium to facilitate zygoteformation.

[0084] Step 3. Transfer zygotes to medium containing geneticin andnourseothricin to select for growth of MATa/α diploid cells.

[0085] Step 4. Transfer MATa/α diploid cells to sporulation medium toinduce spore formation.

[0086] Step 5. Transfer spores to synthetic medium lacking histidine andcontaining canavanine to select for growth MATa haploid spore-progeny.

[0087] Step 6. Transfer MATa haploid spore-progeny to medium containinggeneticin and nourseothricin to score for growth and viability of MATabni1Δ::nat xxxΔ::kan double-mutants.

[0088] In step 2, yeast cells are arrayed on rich medium to allowefficient mating. In step 3, the mating reactions are arrayed ontomedium containing geneticin and nourseothricin, which allows forselected growth of diploid cells. In step 4, the diploid cells aretransferred to medium that is low in nitrogen and carbon and inducessporulation. In step 5, the spores are transferred to germination mediumthat selects for growth of the haploid MATa cells (this step isdescribed in detail below). In step 6, double mutant strains areselected for growth the two mutations are scored as syntheticlethal/fitness defect if the MATa haploid double-mutants form a colonythat is smaller than that associated with either of the single mutants.The mutations predicted to be synthetically lethal can be analyzed inmore detail through tetrad analysis of the heterozygous diploid cellscreated in Step 2.

Example 2 Use of Robotics to Generate Output Array

[0089] Following construction of a natR-marked mutant for syntheticlethal analysis, simple replica plating or pinning manipulations willenable us to complete steps 2-6 of Example 1. These steps can be carriedout through the use of a robotic colony arrayer, as described below. TheColony Arrayer used in this Example, the Virtek Vision CPCA was designedby our labs in conjunction with Virtek Vision. The CPCA is based upon asystem used for genome-wide two-hybrid arrays, since the manipulationsrequired for automated two-hybrid screens are very similar to thoserequired for automated double-mutant construction and synthetic lethalscreens.

[0090] For a rapid genome-wide two-hybrid screening procedure, we havecreated a robotic colony array which includes replica-plate pinhead thattransfers 768 individual colonies from one standard microtiter-sizedagar-slab plate to another in a single move. This cell density allowsefficient mating, on the order of hundreds of zygotes formed per spotpinned, a frequency that will easily suffice for genome-wide syntheticlethal screens.

[0091] The general specifications of a colony arrayer are as follows:

[0092] 1. 16 input and 16 output plates;

[0093] 2. Replicating Pinning Head with 96, 384, or 768 pins, for highspeed transfer of the colonies of different array density;

[0094] 3. A gripper for handling the plates, and if desired forremoving/replacing the covers for full automation;

[0095] 4. Wash/Dry station for cleaning the pins between runs andincludes the 5 stages of water, ethanol or bleach, sonicator (water),ethanol, and air-drying;

[0096] 5. Controlled environment enclosure, with HEPA filter,humidifier, positive pressure and internal UV lamps to ensure fullsterilization of the environment before arraying process.

Example 3 Recovery of Haploid Spore Progeny

[0097] The recovery of haploid spore progeny is mentioned in step 5 inExample 1, and is described in greater detail below. The MATα startingstrain described in Example 1, Y2454, carries two selectable markers,can1Δ0 and MFA1pr-HIS3, both of which permit efficient recovery ofhaploid spore progeny.

[0098] i) MFA1pr-HIS3

[0099] MFA1 encodes the a-factor precursor, which is expressedconstitutively in MATa cells. The MFA1 promoter, MFA1pr, is repressed inMATα and MATa/α cells. The MFA1pr-HIS3 reporter was constructed byreplacing the MFA1 ORF (open reading frame) with the HIS3 ORF such thatMFA1pr drives HIS3 expression. The MATα starting strain, Y2454, fails togrow on synthetic medium lacking histidine because the MFA1pr-HIS3reporter is repressed in MATα cells. The cells constructed in step 2 ofExample 1 will also fail to grow on synthetic medium lacking histidinebecause the MFA1pr-HIS3 reporter is repressed in MAT a/α cells.Following sporulation of the diploid cells in Step 4 of Example 1, theMFA1pr-HIS3 reporter selects for growth of MATa haploid progeny.Twenty-five per cent of the haploid progeny generated by sporulation ofthe diploid cells will be MATa MFA1pr-HIS3. Other a-specific reporterscan be constructed using promoters from different a-specific genes (e.g.MFA2, ASG7, STE2) or different reporters (e.g. URA3, LEU2, orheterologous genes conferring resistance to antibiotics or otherchemicals)

[0100] ii) can1Δ0

[0101] The CAN1 gene encodes an arginine permease. The Y2454 startingstrain has been engineered to carry a recessive can1Δ0 null allele,which renders the cells resistant to canavanine, a toxic arginine analogthat is transported by the CAN1 gene product. The knock-out strainsconstructed by the deletion consortium are canavanine-sensitive becausethey carry a wild-type CAN1 locus. The MATα starting strain, Y2454,carries can1Δ0 rendering it canavanine-resistant. The MATa/α can1Δ0/CAN1diploids isolated in step 3 of Example 1 will be canavanine-sensitive.Following sporulation of the diploid cells in step 4 of Example 1, thecan1Δ0 allele allows for selection of canavanine-resistant haploidprogeny. Fifty percent the haploid spore progeny will becanavanine-resistant. Other recessive drug resistant genes, such as cyh2mutations which leads to cycloheximide resistance, can also be used.

[0102] Efficient Selection for Haploid Spore Progeny

[0103] Both the can1Δ0 and the MFA1pr-HIS3 reporter allow selection forhaploid spore progeny in Step 5 of the pinning procedure described inExample 1. The selection provided by the MFA1pr-HIS3 reporter enablesthe specific isolation of MATa cells. It is important to isolate sporeprogeny of a single mating type when ultimately scoring for the presenceor absence of two marked mutant alleles; otherwise progeny of oppositemating type may conjugate to generate diploid cells heterozygous foreach mutation, which would appear, perhaps falsely, as a viabledouble-mutant.

[0104] Initially, we tested if the MFA1pr-HIS3 reporter would sufficefor isolation of MATa haploid spore progeny in step 5. These testsrevealed that, prior to incubation on sporulation medium, a fraction ofthe MATa/α MFA1-HIS3/MFA1 cells became competent for growth on mediumlacking histidine. This process appears to involve mitotic recombinationbetween the centromere and the MAT locus on chromosome III, creatingMATa/a MFA1-HIS3 cells, which express MFA1-HIS3 and grow on mediumlacking histidine. In a synthetic lethal screen, these MATa/a diploidcells would be heterozygous for the NAT-marked and the KAN-markedalleles and appear as viable double-mutant haploid cells. To overcomethis problem, we introduced a can1Δ0 mutation into our starting strain;can1 mutant alleles have been used extensively to select forcanavanine-resistant haploids in random spore analysis. Analogously, theMATa/α can1Δ0/CAN1 MFA1pr-HIS3/MFA1 diploids, constructed in steps 2 and3, can become canavanine-resistant as diploid cells through mitoticrecombination involving the can1Δ0 locus, creating MATa/α can1Δ0/can1Δ0MFA1pr-HIS3/MFA1 cells. Importantly, prior to incubation on sporulationmedium, we do not observe the formation of cells that are bothcanavanine-resistant and competent for growth on medium lackinghistidine. Thus, the frequency of these two distinct mitoticrecombination events is below the level of detection and we will notobserve MATa/a can1Δ0/can1Δ0 MFA1pr-HIS3/MFA1 cells as background duringa synthetic lethal screen.

Example 4 Genetic Exceptions

[0105] 4A. xxxΔ::kanR Deletion Mutations Linked to MFA1pr-HIS3 or can1Δ

[0106] While the MFA1pr-HIS3 and the can1Δ alleles are essential forsynthetic lethal analysis via a pinning procedure, they present aposition-based problem. The xxxΔ::KANR deletion mutations that arelinked tightly to these alleles will be paired into the genome of thehaploid spores at reduced frequency and may lead to a false syntheticlethal score. However, because the position of every ORF is definedprecisely, the problematic double-mutant combinations are predictable.For mutations in the vicinity of MFA1pr-HIS3 on chromosome X, we willemploy an MFA2pr-HIS3 starting strain. The MFA2 gene encodes a secondcopy of the a-factor structural gene. Like MFA1pr, MFA2pr leads toa-specific gene expression and will facilitate selection for MATaspores. Because 1 cM in S. cerevisiae is roughly equivalent to 1.5 Kb ofDNA sequence and the average S. cerevisiae gene is approximately 2 Kb,we anticipate that 10-30 genes on either side of the MFA1 locus willhave to be mated to a starting strain containing MFA2pr-HIS3.Alternatively the MFA1pr-HIS3 could be moved to another position in thegenome. The can1Δ0 allele presents a slightly different problem becauseit cannot be moved like the MATα-specific reporter. To solve thisproblem, we will directly introduce the can1Δ allele into approximately10-30 of the xxxΔ::kanR deletion mutants on either side of the CAN1locus. Alternatively, we could employ another recessive drug resistantmarker, e.g. cyh2.

[0107] 4B. Genetic Interactions with Selection Genes

[0108] Some xxxΔ::kanR deletion mutations may cause synthetic lethalityin synthetic lethal starting strains carrying the MFA1pr-HIS3 and can1Δalleles. Because MFA1 is a specialized gene devoted to conjugation, itis unlikely that MFA1pr-HIS3 will be associated with any syntheticlethal interactions unless localized alterations of the genome affectthe function of neighboring genes. Because the CAN1 gene productfacilitates arginine uptake, the can1Δ deletion mutation will besynthetically lethal with any gene that prevents arginine biosynthesis.All the genes required for arginine biosynthesis will bedefined/confirmed simply by creating all the double mutants with thewild-type version of the starting strain.

[0109] 4C. Defects in Mating and Spore Formation

[0110] Some xxxΔ::KANR deletion mutations will be defective for one ofthe cellular functions required for double mutant construction or sporeformation. For example, mating defective mutants (e.g. ste4Δ) will failto form diploids in Step 2 of the pinning procedure. A genome-widesynthetic lethal screen will also identify all the genes required forsporulation in Step 4. Obviously, diploids that are homozygous forcertain mutations will lead to a sporulation defect; however, othergenes may prove haploinsufficient for sporulation or two mutations mayexhibit nonallelic-noncomplementation, giving rise to a sporulationdefect. To distinguish the mutants defective for these functions,analysis of the growth of MATa MFA1pr-HIS3 can1Δ cells in Step 5 will beimportant.

Example 5 Comprehensive Synthetic Lethal Screen in Yeast

[0111] A large-scale comprehensive synthetic lethal analysis can beperformed either by constructing approximately 5,000 gene deletions inthe synthetic lethal starting strain, Y2454, or by establishing anautomated method to transfer the knockout alleles constructed by thedeletion consortium into the Y2454 starting strain. In this example, weutilize an automated introduction of the deletion consortium knockoutalleles into a synthetic lethal starting strain. First we switch eachkanR-marked allele to a natR-marked allele and then select for thepresence of MFA1pr-HIS3 in the MATα synthetic lethal starting strainfollowing a genetic cross and a series of pinning steps.

[0112] Switching the kanR-marked allele to a natR-marked allele isachieved easily through transformation of xxxΔ::KAN strains with akanR-natR switcher-cassette, in which the natR gene has been engineeredso that it is flanked by sequences within the original kanR disruptioncassette, and subsequent screening of the nat-resistance transformantsfor kanamycin sensitivity. Strain growth and transformation will becarried out using a 96 well format; therefore, approximately 52 roundsof transformation will allow us to switch the panel of approximately5000 viable MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 xxxΔ::kan strains to MATαura3Δ0 leu2Δ0 his3Δ1 met15Δ0 xxxΔ::nat strains.

[0113] The next challenge is to move the approximately 5,000 xxxΔ::natalleles from the MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 xxxΔ::nat cells intothe strain that carries the haploid selection alleles, MFA1pr-HIS3 andcan1Δ0. Of the alleles specific for the MATα strain, lys2Δ0 and can1Δ0are associated with drug-mediated selections; however, we must constructa haploid selection marker that is functionally equivalent toMFA1pr-HIS3 and selects specifically for growth of MATα cells.

[0114] Construction of two tightly linked reporters, one that selectsfor MATa cells and another that selects for MATα cells, provides asolution to this problem. A dual reporter, MFA1pr-HIS3::MFα1pr-LEU2,will be created simply by integrating MFα1pr-LEU2 just downstream ofMFA1pr-HIS3. As described above, the MFA1pr-HIS3 reporter provides aselection for haploid MATa cells on medium lacking histidine. MFα1prcontrols the expression of the α-factor structural gene and is expressedonly in MATα cells. MFα1pr-LEU2 provides a selection for MATα cells onsynthetic medium lacking leucine. Because MFA1pr-HIS3 is tightly linkedto MFα1pr-LEU2 within the context of the dual reporter, the MATα cellsrecovered on synthetic medium lacking leucine will also carryMFA1pr-HIS3. Several variations of this theme are possible. For example,the MFA1pr-HIS3 could be placed at CAN1 locus, creatingcan1Δ0::MFA1pr-HIS3; in this context, growth of MATα MFα1pr-LEU2can1Δ0::MFA1pr-HIS3 cells can be selected on synthetic medium containingcanavanine and lacking leucine.

[0115] Experimental Steps for a 5,000×5,000 Genome-wide Synthetic LethalScreen

[0116] Step 1. 96-well format transformation will be used to switch apanel of approximately 5000 viable MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0xxxΔ::kanR strains to MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 xxxα::NATstrains. Transformants that grow on medium containing nourseothricinwill be screened for Geneticin sensitivity to confirm the switchingevent.

[0117] Step 2. The MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 xxxΔ::natR strainswill be mated to MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0MFA1pr-HIS3::MFα1pr-URA3 can1Δ0 on rich medium. The resultant diploidcells will be selected for growth on synthetic medium lacking methionineand lysine. As mentioned above, this selection is not ideal because themet15Δ0 allele does not completely eliminate growth on medium lackingmethionine; however, because each strain will only require mating once,we will be able to follow the diploid selection carefully usingrelatively large patches of cells and double replica plating toselective medium.

[0118] Step 3. Sporulation of the resultant diploid cells and selectionfor MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0 MFA1pr-HIS3::MFα1pr-URA3 can1ΔxxxΔ::LEU2 on medium that lacks uracil and leucine but containsα-aminoadipate and canavanine. α-aminoadipate selects for lys2Δ0 mutantcells and canavanine selects for can1Δ0 cells; 50% of the resultanthaploids will be met15Δ0. The presence of the met15 marker can be scoredby successive replica-platings to medium lacking methionine.

[0119] Step 4. Each of the approximately 5000 MATα ura3Δ0 leu2Δ0 his3Δ1lys2Δ0 MFA1pr-HIS3::MFα1pr-URA3 can1Δ xxxΔ::LEU2 strains will each bemated to the array of MATa xxxΔ::KANR deletion mutants and run throughsteps 2-6 of the pinning procedure outlined in Example 1 for systematicgenome-wide synthetic lethal analysis.

Example 6 Input Array of 5,000 Strains Crossed with Particular Mutation

[0120] In a large-scale application of the synthetic genetic analysis ofthe present invention, we screened a bni1Δ query mutation against anarray of 4,644 different viable deletion strains. The array waspropagated on a set of agar plates at a density of 384 strains perplate. For screening, the array was first pinned manually andsubsequently adapted for high-throughput automation using robotics thatwe designed specifically for the manipulation of high density yeastarrays. To ensure reproducibility within a screen and to facilitatevisual scoring, each gene deletion strain was arrayed in pairs, at 768strains per plate. We scored 67 potential synthetic lethal/sickinteractions, 51 (76%) of which were confirmed by tetrad analysis. Togroup the identified genes by functional classification, we assembled alist of their cellular roles defined by the Yeast Proteome Database. Asshown in Table 1, these interactions were highly enriched for genes withroles in cell polarity (20%) cell wall maintenance (18%), and mitosis(16%). Pathways critical for the fitness of bni1Δ cells were revealed bymultiple interactions with subsets of genes involved in bud emergence(BEM1, BEM2, and BEM4), chitin synthase III activity (CHS3, SKT5, CHS5,CHS7, and BNI4), MAP kinase pathway signaling (BCK1 and SLT2), the cellcycle-dependent transition from apical to isotropic bud growth (CLA4,ELM1, GIN4, and NAP1), and the dynein/dynactin spindle orientationpathway (DYN1, DYN2, PAC1, PAC11, ARP1, JNM1, NIP100). Importantly, weidentified 8 of the 10 previously known bni1Δ synthetic lethal/sickinteractions, which include BNR1, HOF1, CDC12, SLT2, BCK1, PKC1, genesimplicated in polarized morphogenesis and cell wall maintenance, andASE1, DYN1, ARP1, NIP100, genes implicated in mitotic spindle function.Of those that were not identified, CDC12 and PKC1 were not contained inour deletion set, while cells lacking HOF1 grow very slowly and areapparently beyond the sensitivity of the assay. In total, we discovered42 novel synthetic genetic interactions for bni1Δ, including 10 genes ofunclassified function.

[0121] Table 1 below lists a set of synthetic lethal/sick interactionsobserved for query mutations in BNI1, ARC40, BBC1, NBP2, BIM1, RAD27,and SGS1. TABLE 1 BNI1 Cell Role BEM1 Cell Polarity BEM2 Cell PolarityBEM4 Cell Polarity BUD6^(†) Cell Polarity SLA1^(†) Cell Polarity CLA4Cell Polarity ELM1^(†) Cell Polarity GIN4 Cell Polarity NAP1^(†) CellPolarity SWE1^(†) Cell Polarity BNR1 Cytokinesis CYK3^(†) CytokinesisSHS1 Cytokinesis BCK1 Cell Wall Maintenance BNI4^(†) Cell WallMaintenance FAB1 Cell Wall Maintenance CHS3 Cell Wall MaintenanceSKT5^(†) Cell Wall Maintenance CHS5^(†) Cell Wall Maintenance CHS7^(†)Cell Wall Maintenance SLT2 Cell Wall Maintenance SMI1^(†) Cell WallMaintenance ARP1 Mitosis ASE1 Mitosis DYN1 Mitosis DYN2^(†) Mitosis JNM1Mitosis NIP100 Mitosis NUM1 Mitosis PAC1 Mitosis ATS1 Cell StructurePAC11 Cell Structure YKE2^(†) Cell Structure PCL1^(†) Cell Cycle ControlDRS2 RNA Processing SNC2 Vesicular Transport VPS28 Vesicular TransportYPT6^(†) Vesicular Transport ELP2 Pol II Transcription ELP3^(†) Pol IITranscription BBC1^(†) Unknown NBP2^(†) Unknown TUS1^(†) UnknownYBL051c^(†) Unknown YBL062w^(†) Unknown YDR149c Unknown YHR111w^(†)Unknown YKR047w^(†) Unknown YLR190w^(†) Unknown YMR299c^(†) UnknownYNL119w^(†) Unknown BBC1 Cell Role BEM1 Cell Polarity BEM4 Cell PolarityBNI1^(†) Cell Polarity SLA1^(†) Cell Polarity CAP1^(†) Cell StructureCAP2^(†) Cell Structure PAC10^(†) Cell Structure GIM3 Cell StructureGIM5 Cell Structure SAC6 Cell Structure CHS5^(†) Cell Wall MaintenanceRAS2 Signal Transduction ELP2^(†) Pol II Transcription ELP3^(†) Pol IITranscription SDS3 Chromatin Structure YLR235c Unknown YML095c-A UnknownARC40 Cell Role ARC18 Cell Polarity BEM1 Cell Polarity BEM2 CellPolarity CLA4 Cell Polarity MYO5^(†) Cell Polarity PEA2^(†) CellPolarity VRP1 Cell Polarity BCK1 Cell Wall Maintenance BNI4^(†) CellWall Maintenance CHS3 Cell Wall Maintenance SKT5 Cell Wall MaintenanceCHS5 Cell Wall Maintenance CHS6 Cell Wall Maintenance CHS7 Cell WallMaintenance HOC1 Cell Wall Maintenance KRE1 Cell Wall Maintenance SLT2Cell Wall Maintenance SPF1^(†) Cell Wall Maintenance YER083c^(†) CellWall Maintenance YKE2 Cell Structure GIM3 Cell Structure GIM4^(†) CellStructure CCT3^(†) Cell Structure SAC6 Cell Structure GLO3 VesicularTransport SAP155^(†) Cell Cycle Control SEC66 Protein Modification ILM1Energy Generation MNN11 Protein Modification STE24^(†) ProteinModification CIK1^(†) Meiosis RIM101^(†) Meiosis RUD3 VesicularTransport SEC22 Vesicular Transport TFP3 Small Molecule TransportCPR7^(†) Protein Folding SHE4 Differentiation SUM1^(†) Pol IITranscription YBL062w Unknown YLR111w^(†) Unknown ARP2 Cell RoleBEM1^(†) Cell Polarity BEM2^(†) Cell Polarity CLA4^(†) Cell PolarityPEA2^(†) Cell Polarity PRK1^(†) Cell Polarity RGD1^(†) Cell PolarityRVS161 Cell Polarity RVS167^(†) Cell Polarity VRP1^(†) Cell PolarityBCK1 Cell Wall Maintenance BNI4 Cell Wall Maintenance CHS3 Cell WallMaintenance SKT5 Cell Wall Maintenance CHS5^(†) Cell Wall MaintenanceCHS6^(†) Cell Wall Maintenance CHS7^(†) Cell Wall Maintenance HOC1 CellWall Maintenance KRE1 Cell Wall Maintenance SLT2 Cell Wall MaintenanceSPF1^(†) Cell Wall Maintenance YER083c Cell Wall Maintenance YKE2 CellStructure PAC10 Cell Structure GIM3 Cell Structure GIM4 Cell StructureSAC6 Cell Structure SAC7^(†) Cell Structure ILM1 Energy GenerationSAP155^(†) Cell Cycle Control SEC66 Protein Modification MNN11 ProteinModification STE24^(†) Protein Modification BTS1 Protein ModificationRUD3 Vesicular Transport CPR7 Protein Folding SHE4^(†) DifferentiationSUM1 Pol II Transcription SRO9 Protein Synthesis UTH1^(†) Aging DEP1Lipid Metabolism YBL062w Unknown YDR018c Unknown YGL250w^(†) UnknownYLR111w^(†) Unknown BIM1 Cell Role ARP1 Mitosis ASE1 Mitosis BIK1Mitosis CHL4 Mitosis DYN1 Mitosis JNM1 Mitosis KIP3 Mitosis NUM1 MitosisPAC1 Mitosis SLK19 Mitosis BFA1 Mitosis BUB1 Mitosis BUB2 Mitosis BUB3Mitosis MAD1 Mitosis MAD2 Mitosis MAD3 Mitosis CSM3 Meiosis MCK1 MeiosisCTF8 Chromatin Structure CTF19 Chromatin Structure DCC1 ChromatinStructure IML3 Chromatin Structure MCM21 Chromatin Structure MCM22Chromatin Structure PHO23 Chromatin Structure SAP30 Chromatin StructureBEM1 Cell Polarity ARP6^(†) Cell Structure GIM3 Cell Structure GIM4 CellStructure GIM5 Cell Structure PAC11 Cell Structure FAB1 Cell WallMaintenance SMI1 Cell Wall Maintenance ELP2 Pol II Transcription INP52Vesicular Transport RAD54 DNA Repair PPZ1 Signal Transduction KEM1 RNAProcessing AOR1 Unknown IES2^(†) Unknown MRC1 Unknown RTT103^(†) UnknownVID22 Unknown YTA7 Unknown YBR095c Unknown YDR149c Unknown YGL211w^(†)Unknown YGL217c Unknown YNL170w Unknown YLR381w Unknown YLR386w UnknownYML095c-A Unknown YPL017c Unknown NBP2 Cell Role BIM1 Mitosis CIN2^(†)Mitosis KAR9 Mitotis KIP3 Mitosis PAC10 Cell Structure GIM5 CellStructure CAP1^(†) Cell Structure CAP2^(†) Cell Structure BNI1^(†) CellPolarity FAB1 Cell Wall Maintenance SMI1^(†) Cell Wall MaintenanceVAM7^(†) Vesicular Transport VPS29^(†) Vesicular Transport RPL16A^(†)Protein Synthesis RPS18B^(†) Protein Synthesis RPS23A^(†) ProteinSynthesis CPR6^(†) Protein Folding FPR1^(†) Protein Folding CLB4^(†)Cell Cycle Control REM50^(†) DNA Repair RTG2^(†) Carbohydrate MetabolismRTG3^(†) Carbohydrate Metabolism MON1^(†) Unknown YDL063c^(†) UnknownYGL211w^(†) Unknown YGL217c Unknown YML095c-A Unknown SGS1 Cell RoleASF1^(†) DNA Repair HPR5 DNA Repair POL32 DNA Repair RAD27 DNA RepairRAD50 DNA Repair SAE2 DNA Repair SLX1 DNA Repair MMS4 DNA Repair MUS81DNA Repair SLX4 DNA Repair WSS1 DNA Repair RNR1 DNA Synthesis RRM3 DNASynthesis YNL218w^(†) DNA Synthesis CSM3^(†) Meiosis ESC2^(†) ChromatinStructure ESC4^(†) Chromatin Structure TOP1^(†) Chromatin StructureSWE1^(†) Cell Cycle Control PUB1^(†) RNA Processing RPL24A^(†) ProteinSynthesis SIS2^(†) Cell Stress SOD1 Cell Stress YBR094w Unknown RAD27Cell Role DDC1^(†) DNA Repair EXO1^(†) DNA Repair HPR5 DNA Repair MRE11DNA Repair MMS4^(†) DNA Repair MUS81^(†) DNA Repair RAD9 DNA RepairRAD17^(†) DNA Repair RAD24^(†) DNA Repair RAD50 DNA Repair RAD51 DNARepair RAD52 DNA Repair RAD54 DNA Repair RAD55 DNA Repair RAD57 DNARepair SAE2 DNA Repair XRS2 DNA Repair CTF4^(†) DNA Synthesis CAC2^(†)Chromatin Structure ESC2^(†) Chromatin Structure HST1^(†) ChromatinStructure HST3^(†) Chromatin Structure HPC2^(†) Pol II TranscriptionCSM3^(†) Meiosis DOC1 Cell Cycle Control RPL27A Protein SynthesisRPS30B^(†) Protein Synthesis YDJ1 Protein Translocation LYS7 Cell StressSIS2^(†) Cell Stress SOD1 Cell Stress FYV11 Unknown YLR352w^(†) UnknownYNL171c Unknown YPR116w Unknown

Example 7 Demonstration that Pinning Procedure Works for SyntheticLethal Analysis

[0122] The present example demonstrates that the replica pinningprocedure of the present invention can be used for synthetic lethalanalysis. A deletion of the BNI1 gene, bni1Δ, was selected as the querymutation. A test-array of gene deletion mutants was assembled thatincluded bnr1Δ, which is synthetically lethal with bni1Δ. BNI1 and BNR1both encode members of the formin family, proteins that appear tocontrol actin polymerization in response to signaling by Rho-typeGTPases. Growing yeast cells contain two major filamentous actinstructures, cortical actin patches, which polarize to the cortex of thegrowing bud and act as sites of endocytosis, and actin cables, whichalign along the mother bud axis and act as tracks for myosin motors thatcoordinate polarized cell growth and spindle orientation. Bni1 and Bnr1are required for the formation of actin cables. bni1Δ mutants showdefects in polarized cell growth and spindle orientation, whereas bnr1Δmutants display no obvious phenotype, indicating that Bni1 functions asthe predominant formin in yeast cells. The array contained 96 strains,each of which was included in quadruplicate and positioned next to eachother in a square pattern, resulting in a matrix with 384 elements.bnr1Δ was included at two positions and enriched the array for mutationsin other genes with roles in actin assembly and cell polarity.

[0123] The array of bni1Δ double-mutants resulting from the finalpinning and the corresponding wild-type control are shown in FIG. 2A. Asanticipated, the cells at the bnr1Δ positions failed to grow, forming aresidual colony with a reduced size relative to the control. Because theresultant double-mutants are created by meiotic recombination, genedeletions that are genetically linked to the query mutation form doublemutants at a reduced frequency. Moreover, when the query mutation isactually identical to one of the gene deletions within the array, doublemutants can not form. Thus, we also anticipated that the cells at thebni1Δ position would fail to grow under double-mutant selection. Novelsynthetic genetic interactions were observed with gene deletionmutations of CLA4 and BUD6 (AIP3). Cla4 is a PAK-like kinase involved inactin patch assembly and the cell cycle-dependent transition from apicalto isotropic bud growth; Bud6 forms a complex with Bni1 and actin tocontrol actin cable assembly and cell polarity. Tetrad analysisconfirmed that both the bni1Δ bnr1Δ and bni1Δ cla4Δ double-mutants wereinviable and that the bni1Δ bud6Δ double-mutant was associated with aslower growth rate or “synthetic sick” phenotype, reflecting reducedfitness of the double-mutant relative to the respective single mutants(FIG. 2B). This example thus demonstrates that the replica pinningprocedure can identify genetic interactions corresponding to thespectrum of fitness defects from synthetic sick to synthetic lethalphenotypes.

Example 8 Input Array of 5,000 Strains Crossed with UncharacterizedQuery Mutation

[0124] If synthetic lethal/sick interactions identify functionallyrelated genes, then some of the uncharacterized genes identified in thebni1Δ screen should also participate in cortical actin assembly and cellpolarity. To test this possibility, we conducted synthetic lethal/sickscreen for a previously uncharacterized gene, BBC1. The bbc1Δ deletionmutant shows no obvious phenotype and the Bbc1 amino acid sequence isnot indicative of a cellular role; however, Bbc1 contains an SH3protein-protein interaction domain, suggesting that Bbc1 may function aspart of a complex. We scored 16 potential synthetic lethal/fitnessinteractions for bbc1Δ, the majority of which have YPD-classified cellpolarity or cell structure (cytoskeletal) roles (Table 1). Inparticular, bbc1Δ showed interactions with several genes whose productscontrol actin polymerization and localize to cortical actin patches(CAP1, CAP2, SAC6, and SLA1), suggesting BBC1 may be involved in theassembly actin patches or their dependent processes. The results ofadditional experiments demonstrated that Bbc1 localized predominantly tocortical actin patches and that its SH3 domain binds directly to Las17(Bee1), a member of the WASP (Wiskott-Aldrich syndrome protein) familyproteins that controls the assembly of cortical actin patches throughregulation of the Arp2/3 actin nucleation complex.

Example 9 Input Array of 5,000 Strains Crossed with Helicase Mutation

[0125] To explore the potential for the synthetic genetic analysis ofthe present invention to identify interactions for genes with rolesdistinct from cytoskeletal organization and cell polarity, we undertookscreens with two non-essential genes that function in DNA damage andrepair pathways. SGS1 encodes the yeast homolog of the human Werner'sSyndrome protein, WRN, a member of the RecQ family of DNA helicases,while RAD27 encodes an enzyme that processes Okazaki fragments duringDNA synthesis and repair. The phenotype of yeast cells deleted for theSGS1 gene mirrors the chromosomal instabilities and premature agingassociated with Werner's syndrome. SGS1 is known to show a syntheticlethal/sick relationship with YNL218W, which encodes the yeast homologof human Werner helicase interacting protein, and seven other genes(SLX1, MMS4, SLX3, SLX4, HEX3, HRP5, and SLX8), which are thought tomediate the resolution of recombination intermediates generated in theabsence of SGS1. In total, we scored 24 potential syntheticlethal/fitness interactions for SGS1, the majority of which wereassociated with cellular roles in DNA synthesis and repair (Table 1). Weidentified 6 of the previously known synthetic lethal/sick interactions;the 2 that were missed (HEX3, SLX8) had severe growth defects thatprecluded propagation through the multiple pinning steps. Several novelSGS1 synthetic lethal/sick interactions are of considerable interest:RRM3, a gene that encodes a closely related helicase involved in rDNAreplication; WSS1, a gene identified as a high dosage suppressor ofSMT3, which codes a conserved ubiquitin-related protein that interactswith HEX3 in the two-hybrid system; YBR094W, a highly conserved gene ofunknown function with an NH2-terminal SurE domain and COOH-terminaltubulin-tyrosine ligase-like domain; and ESC4, which codes for anuncharacterized gene with 3 BRCT domains, peptide recognition modulesthat appear to be found exclusively in proteins involved in DNAsynthesis and repair.

Example 10 Network of Synthetic Lethal Analysis

[0126] Because many of the synthetic lethal/sick relationships appear toinvolve functionally related genes, the data set derived fromlarge-scale synthetic genetic interactions should form a highlyconnected network in which genes are classified base upon theirconnectivities. To build a network of interactions and further explorethe potential of this methodology, we conducted additional syntheticlethal/sick screens with specific query mutations in the followinggenes:

[0127] (i) bim1Δ, a complete deletion of BIM1, whose product localizesto the tips of astral microtubules and controls the orientation of themitotic spindle.

[0128] (ii) nbp2Δ, a complete deletion of NBP2, gene of uncharacterizedfunction that showed a synthetic lethal/sick interaction with BNI1; theNbp2 product contains an SH3 domain and shows a two-hybrid interactionwith Nap1.

[0129] (iii) arc40-1, a temperature sensitive allele of ARC40, anessential gene that codes for a component of the Arp2/3 complex, aseven-member complex that functions to nucleate actin filaments,controlling the assembly, movement, and localization of cortical actinpatches in yeast.

[0130] (iv) arp2-2, a temperature sensitive allele of ARP2, coding forone of the key actin-related proteins of the Arp2/3 complex.

[0131] (v) rad27Δ, a complete deletion of RAD27, which showed asynthetic lethal/sick interaction with SGS1 and whose product functionsas a nuclease that processes Okazaki fragments during DNA synthesis.

[0132] To plot the resultant synthetic lethal/sick interactions as anetwork, the data set was first imported into the BiomolecularInteraction Network Database (BIND), then formatted with BIND tools andexported to the Pajek package, a program originally designed for theanalysis of social networks. The genetic interaction network shown inFIG. 3 contains 205 genes, represented as nodes on the graph, 292synthetic lethal/sick interactions, represented as edges connectinggenes. All of these interactions were first identified using theautomated methodology and then confirmed by tetrad analysis. Tovisualize sets of genes with related functions, we color-coded the genesaccording to their YPD cellular roles and aligned the genes based uponboth their roles and connectivity. These relationships can also berepresented by two-dimensional hierarchical clustering, as is used foranalysis of DNA microarray experiments. For genetic interaction data,both the query genes and the interacting genes are clustered based uponthe similarity of their patterns of genetic interactions (FIG. 4).

[0133] Each of the query genes were biased towards interactions withgenes of particular cellular roles. Moreover, subsets of interactinggenes with the same cellular roles could distinguished from one anotherby their connectivity. For example, the BIM1 screen identified a largegroup of genes involved in mitosis (red genes), which include severalcomponents of the Bub2p- and Mad2p-dependent spindle assemblycheckpoints (BUB1, BUB2, BUB3, BFA1, MAD1, MAD2, and MAD3) and multiplegenes involved nuclear migration and spindle orientation during mitosis(BIK1, MCK1, SLK19, KIP3, PAC11, PAC1, NUM1, DYN1, JNM1, ARP1, ASE1), asubset of which also interacted with BNI1 and function specifically aspart of the Dyn1 kinesin pathway. In addition, BIM1 interacted with agroup genes that have a chromatin/chromosome structure cellular role(yellow genes), many of which have been implicated kinetichore function(CTF8, CTF19, MCM21, MCM22, and CHL4), and a total of 15 genes of withunknown cellular roles (black genes). To examine one of theseuncharacterized genes in more detail, we conducted an NBP2 screen, whichshowed interactions with several genes involved in nuclear migration andspindle function (KAR9, CIN2 and KIP3), actin assembly (CAP1 and CAP2),and de novo folding of actin and tubulin (PAC10 and GIM5), suggestive ofa general role in cytoskeletal organization.

[0134] The RAD27 screen resulted in 35 interactions, the majority ofwhich occurred for genes with DNA synthesis/repair cellular roles (FIG.3 green genes). The unprocessed Okazaki fragments of rad27Δ cells areprobably recognized as nicks or converted into double-strand breaks asevidenced by a large set of previously known synthetic lethal/sickinteractions with the genes encoding multiple components of therecombinational repair apparatus (RAD50, RAD51, RAD52, RAD54, RAD55,RAD57, MRE11, XRS2) and the DNA damage checkpoint signaling pathway(RAD9, RAD17, RAD24, DDC1). Intriguingly, we observed novel interactionswith HST1 and HST2, two genes encoding Sir3-like deacetylases and CAC2,a gene coding for a chaperone that delivers acetylated histones to newlysynthesized DNA, which may be indicative of a functional relationshipamongst the products of these genes in chromatin assembly or silencing.RAD27 also showed a synthetic lethal/fitness with the SOD1 superoxidedismutase gene, and its copper chaperone, LYS7, suggesting that theantioxidant functions of Sod1 are required to protect the rad27Δ cellsfrom accumulated DNA damage. Finally, RAD27 interacted with 4 genes ofunknown function, for which we predict a possible role in a DNAsynthesis and/or repair.

[0135] We scored 40 synthetic lethal/sick interactions for arc40-1, mostof which have cell polarity or cell wall maintenance roles (Table 1),including 2 genes involved in Arp2/3 activation (VRP1 and MYO5). From asimilar screen with a temperature sensitive ARP2 allele, arp2-1, wescored a total of 43 synthetic lethal/sick interactions. Strikingly, theARP2 screen identified a large subset of 32 interactions that wereshared with ARC40. Both screens identified a relatively small number ofunique interactions, which included genes implicated in actin patchassembly (MYO5 with arc40-1 but not arp2-1; ARC18 and PRK1 with arp2-1but not arc40-1) and may reflect roles specific to each Arp2/3 subunit.These findings suggest that genes whose products form functionalcomplexes will show a highly similar but unique set of geneticinteractions.

Example 11 Plasmid-borne Approaches

[0136] The pinning procedure described for the construction of doublemutants provides a simple method to move a plasmid of interest into theset of approximately 5,000 viable xxxΔ::KAN deletion mutants. In thisprocedure, the Y2454 starting strain is transformed with a URA3- orLEU2-based plasmid and then crossed into the haploid mutants byfollowing steps 1-6 of the pinning procedure as described in Example 1for genome-wide double-mutant construction. The ability to undertakeplasmid-based screens with the complete deletion set greatly extends thenumber of possible genome-wide screens including synthetic dosagelethality and green-fluorescence protein-based reporter screens.

Example 12 Synthetic Lethal/Fitness Analysis via Bar-coded DeletionMutants and DNA Microarray Technology

[0137] Genetic interactions for yeast mutant strains that contain “barcodes” for identification can be analyzed using DNA microarrays.

[0138] Step 1. The pool of MATα xxxΔ::KAN cells, containing the entireset of approximately 5,000 viable deletion mutants, will be mated to aY2454-derivative for synthetic lethal analysis with a mutant allelemarked with NAT (e.g. Y2454 made bni1Δ::NAT).

[0139] Step 2. The resultant pool of diploid cells is transferred tosporulation medium and MATa can1Δ MFA1-HIS3 cells are selected onsynthetic medium that contains canavanine but lacks histidine.

[0140] Step 3. The pool of haploid MATa can1Δ MFA1-HIS3 cells are grownon medium containing geneticin to select for MATa can1Δ MFA1-HIS3xxxΔ::KAN deletion mutants

[0141] Step 4. The MATa can1Δ MFA1-HIS3 xxxΔ::KAN cells are split intotwo samples. To determine the set of mutant cells that mated andsporulated efficiently, DNA will be prepared from one sample and used toprobe the bar-coded microarray. DNA preparation of the DNA involvesisolating genomic DNA and preparing bar code probes via a PCR-basedmethod.

[0142] Step 5. To determine the set of synthetic lethal double mutants,the other sample of cells will first be grown on medium containingnourseothricin, which selects for double-mutant cells, e.g. MATabni1Δ::LEU2 xxxΔ::KANR can1Δ MFA1-HIS3 cells. Then, DNA will preparedfor bar-coded microarray analysis. Comparison of the bar-coded mutantsthat are present in sample 1 but not present in sample 2 identifiespotential synthetic-lethal combinations.

[0143] Other Embodiments

[0144] While the invention has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

What is claimed is:
 1. A high density output array of multiple yeaststrains, wherein each resulting yeast strain in the output arraycontains at least two resulting genetic alterations, and wherein theresulting genetic alterations are different in each resulting yeaststrain, the output array being the mating product of at least two inputarrays, wherein at least one of the input arrays comprises multiplestarting strains of yeast, wherein each starting yeast strain carries atleast one genetic alteration, with the genetic alteration beingdifferent in each starting yeast strain.
 2. The output array of claim 1,wherein the resulting yeast strains are in the diploid state.
 3. Theoutput array of claim 1, wherein the resulting yeast strains are in thehaploid state.
 4. The input or output array of claim 1, wherein thestarting and resulting yeast strains are selected from any yeast strainthat has two mating types and is capable of meiotic and mitoticreproduction.
 5. The input or output array of claim 4, wherein thestarting and resulting yeast strains are from either the Saccharomycescerevesiae or the Schizosaccharomyces pombe species.
 6. The input oroutput array of claim 1, wherein the yeast strains are located onplates, with between about 9 and about 6200 yeast colonies on one plate.7. The output array of claim 1, wherein the resulting genetic alterationis a double mutant, the double mutant involving a mutation of twodifferent endogenous yeast genes.
 8. The output array of claim 7,wherein the double mutant carries the deletion of two differentnon-essential yeast genes.
 9. The output array of claim 8, wherein thedouble mutant is either a synthetic lethal double mutant or a syntheticfitness double mutant.
 10. The output array of claim 1, which comprisesbetween about 1,000 and about 25 million resulting strains of yeast. 11.The input array of claim 1, wherein the starting genetic alteration inat least one starting yeast strain is selected from the group consistingof introduction of genes coding for an aptamer, introduction of aprotein-protein interaction detection system, expression of aheterologous gene from a viral, prokaryotic, or eukaryotic genome, withthe heterologous gene either having or not having a yeast homolog,transfection with a promoter operably linked to a reporter gene, andmutation or deletion of an endogenous yeast gene.
 12. The input array ofclaim 11, wherein the aptamer is either a peptide aptamer or a nucleicacid aptamer.
 13. The input array of claim 11, wherein the aptamerperforms a function selected from the group consisting of inhibitingexpression of a gene, increasing expression of a gene, inhibitingprotein-protein interactions, enhancing protein-protein interactions,inhibiting the activity of a protein, and enhancing the activity of aprotein.
 14. The input array of claim 11, wherein the protein-proteininteraction detection system is selected from the group consisting of ayeast two-hybrid system, the Ras recruitment system, the split ubiquitinsystem, and protein fragment complementation systems.
 15. The inputarray of claim 11, wherein the heterologous gene is a human gene. 16.The input array of claim 15, wherein the human gene comprises a set ofalleles, each differing by one or more SNPs.
 17. A method for generatinga high-density output array of resulting multiple yeast strains, whereineach resulting yeast strain carries at least two resulting geneticalterations, and wherein the resulting genetic alterations are differentin each yeast strain, the method comprising: a) generating multiplestarting yeast strains, each strain carrying a starting geneticalteration; b) mating sets of two starting yeast strains, wherein eachof the two starting yeast strains contains a starting geneticalteration; and c) recovering multiple diploid yeast strains which carrya resulting genetic alteration, wherein the resulting genetic alterationcomprises the starting genetic alterations from each of the two matedstarting yeast strains; and d) arraying the genetically altered yeaststrains in a high-density diploid output array.
 18. A method forgenerating a high-density output array of resulting multiple yeaststrains, wherein each resulting yeast strain carries at least tworesulting genetic alterations, and wherein the resulting geneticalterations are different in each yeast strain, the method comprising:a) generating multiple starting yeast strains, each strain carrying astarting genetic alteration; b) mating sets of two starting yeaststrains, wherein each of the two starting yeast strains contain astarting genetic alteration; c) causing the mated strains to undergosporulation, resulting in haploid strains; d) germinating a singlemating type; e) growing the haploid spore progeny using selective growthcriteria; f) recovering multiple haploid yeast strains which carry aresulting genetic alteration, wherein the resulting genetic alterationcomprises the starting genetic alterations from each of the two matedstarting yeast strains; and g) arraying the genetically altered yeaststrains in a high-density format on an output array.
 19. The method ofclaim 18, wherein the starting and resulting yeast strains are selectedfrom any yeast strain that has two mating types and is capable ofmeiotic and mitotic reproduction.
 20. The method of claim 19, whereinthe starting and resulting yeast strains are from either theSaccharomyces cerevesiae or the Schizosaccharomyces pombe species. 21.The method of claim 18, wherein the starting and resulting yeast strainsare located on plates, with between about 90 and 6200 yeast colonies onone plate.
 22. The method of claim 18, wherein the resulting geneticalteration is a double mutant, the double mutant involving a mutation oftwo different endogenous yeast genes.
 23. The method of claim 22,wherein the double mutant carries the deletion of two differentnon-essential yeast genes.
 24. The method of claim 22, wherein thedouble mutant is either a synthetic lethal double mutant or a syntheticfitness double mutant.
 25. The method of claim 18, wherein the outputarray comprises between about 1,000 and about 25 million resulting yeaststrains.
 26. The method of claim 18, wherein the starting geneticalteration in at least one starting yeast strain is selected from thegroup consisting of introduction of genes coding for expression of anaptamer, a protein-protein interaction detection system, expression of aheterologous gene from a viral, prokaryotic, or eukaryotic genome, withthe heterologous gene either having or not having a yeast homolog,transfection with a promoter operably linked to a reporter gene, andmutation or deletion of an endogenous gene.
 27. The method of claim 26,wherein the starting genetic alteration in both starting yeast strainsis selected from the group consisting of introduction of genes codingfor expression of an aptamer, a protein-protein interaction detectionsystem, expression of a heterologous gene from a viral, prokaryotic, oreukaryotic genome, with the heterologous gene either having or nothaving a yeast homolog, transfection with a promoter operably linked toa reporter gene, and mutation or deletion of an endogenous gene.
 28. Themethod of claim 26, wherein the aptamer is either a protein aptamer or anucleic acid aptamer.
 29. The method of claim 26, wherein the aptamerperforms a function selected from the group consisting of inhibitingexpression of a gene, increasing expression of a gene, inhibitingprotein-protein interactions, enhancing protein-protein interactions,inhibiting the activity of a protein, and enhancing the activity of aprotein.
 30. The method of claim 26, wherein the protein-proteininteraction detection system is selected from the group consisting of ayeast two-hybrid system, the Ras recruitment system, the split ubiquitinsystem, and protein fragment complementation systems.
 31. The method ofclaim 26, wherein the heterologous gene is a human gene.
 32. The methodof claim 31, wherein the human gene comprises a set of alleles, eachdiffering by one or more SNPs.
 33. The method of claim 18, wherein thestarting yeast strains carry selectable markers to permit efficientrecovery of haploid spore progeny.
 34. The method of claim 33, whereinthe selectable markers are mating type specific promoters which permitselection of particular haploid mating types.
 35. The method of claim18, wherein robotic manipulation is utilized.
 36. A method forconducting synthetic lethal analysis of yeast colonies within a highdensity array of multiple yeast strains, the method comprising: a)generating a high-density output array of multiple yeast strainsaccording to the method of claim 18; b) comparing the phenotype of thehaploid strains recovered in step f) of claim 18 to the phenotype of thestarting yeast strains; and c) detecting which haploid strains containsynthetic modulations by observing differences in the phenotype of thehaploid strains as compared to the phenotype of the starting strains.37. The method of claim 36, wherein the starting and resulting yeaststrains are selected from any yeast strain that has two mating types andis capable of meiotic and mitotic reproduction.
 38. The method of claim37 wherein the starting and resulting yeast strains are from either theSaccharomyces cerevesiae or Schizosaccharomyces pombe species.
 39. Themethod of claim 36, wherein the starting and resulting yeast strains arelocated on plates, with between about 90 and 6200 yeast colonies on oneplate.
 40. The method of claim 36, wherein the resulting geneticalteration is a double mutant, the double mutant including the mutationof two different endogenous yeast genes.
 41. The method of claim 40,wherein the double mutant carries the deletion of two differentnon-essential yeast genes.
 42. The method of claim 40, wherein thedouble mutant is either a synthetic lethal double mutant or a syntheticfitness double mutant.
 43. The method of claim 36, wherein the outputarray comprises between about 1,000 and about 25 million resulting yeaststrains.
 44. The method of claim 36, wherein the starting geneticalteration in at least one starting yeast strain is selected from thegroup consisting of introduction of genes coding for expression of anaptamer, a protein-protein interaction detection system, expression of aheterologous gene from a viral, prokaryotic, or eukaryotic genome, withthe heterologous gene either having or not having a yeast homolog,transfection with a promoter operably linked to a reporter gene, andmutation or deletion of an endogenous gene.
 45. The method of claim 44,wherein the starting genetic alteration in both starting yeast strainsis selected from the group consisting of introduction of genes codingfor expression of an aptamer, a protein-protein interaction detectionsystem, expression of a heterologous gene from a viral, prokaryotic, oreukaryotic genome, with the heterologous gene either having or nothaving a yeast homolog, transfection with a promoter operably linked toa reporter gene, and mutation or deletion of an endogenous gene.
 46. Themethod of claim 44, wherein the aptamer is either a protein aptamer or anucleic acid aptamer.
 47. The method of claim 44, wherein the aptamerperforms a function selected from the group consisting of inhibitingexpression of a gene, increasing expression of a gene, inhibitingprotein-protein interactions, enhancing protein-protein interactions,inhibiting the activity of a protein, and enhancing the activity of aprotein.
 48. The method of claim 44, wherein the protein-proteininteraction detection system is selected from the group consisting of ayeast two-hybrid system, the Ras recruitment system, the split ubiquitinsystem, and protein fragment complementation systems.
 49. The method ofclaim 44, wherein the heterologous gene is a human gene.
 50. The methodof claim 49, wherein the human gene comprises a set of alleles, eachdiffering by one or more SNPs.
 51. The method of claim 36, wherein thestarting yeast strains carry selectable markers to permit efficientrecovery of haploid spore progeny.
 52. The method of claim 51, whereinthe selectable markers are mating type specific promoters which permitselection of particular haploid mating types.
 53. The method of claim36, wherein robotic manipulation is utilized.
 54. A method for assigninggene function by performing the synthetic lethal analysis of claim 36 togenerate a synthetic lethal profile for a particular double mutant, andthen performing cluster analysis of a set of synthetic lethal profilesto identify mutant alleles that result in similar compromised stateswith similar cellular functions perturbed.
 55. The method of claim 36,wherein the synthetic modulation of the haploid strains is identifiedthrough the use of a genetic tag.
 56. The method of claim 55, whereinthe genetic tag is a unique 20 mer oligonucleotide sequence.
 57. Amethod for conducting synthetic lethal analysis of yeast colonies withina high density output array of multiple resulting yeast strains, themethod comprising: a) generating a high-density output array of multipleyeast strains according to the method of claim 17; b) comparing thephenotype of the diploid strains recovered in step c) of claim 17 to thephenotype of the starting yeast strains; c) detecting which diploidstrains contain synthetic modulations by observing differences in thephenotype of the diploid resultant strains as compared to the phenotypeof the starting strains.
 58. A method for conducting small moleculescreening of yeast colonies within a high density input array ofmultiple starting yeast strains, the method comprising: a) generatingmultiple starting yeast strains in an input array, each strain carryinga starting genetic alteration; b) exposing the starting strains in theinput array to a biological effector; and c) detecting which startingstrains contain synthetic modulations by observing differences betweenthe phenotype of the starting strains before and after exposure to thebiological effector.
 59. The method of claim 58 wherein the biologicaleffector is a small molecule.
 60. The method of claim 59, furthercomprising the step of screening for small molecules which kill yeaststrains carrying a specified deletion mutation.
 61. The method of claim58, wherein the starting yeast strains are selected from any yeaststrain that has two mating types and is capable of meiotic and mitoticreproduction.
 62. The method of claim 61, wherein the starting yeaststrains are from either the Saccharomyces cerevesiae orSchizosaccharomyces pombe species.
 63. The method of claim 58, whereinthe starting yeast strains are located on plates, with between about 90and about 6200 yeast colonies on one plate.
 64. The method of claim 58,wherein the starting genetic alteration in at least one starting yeaststrain is selected from the group consisting of introduction of genescoding for expression of an aptamer, a protein-protein interactiondetection system, expression of a heterologous gene from a viral,prokaryotic, or eukaryotic genome, with the heterologous gene eitherhaving or not having a yeast homolog, transfection with a promoteroperably linked to a reporter gene, and mutation or deletion of anendogenous gene.
 65. The method of claim 64, wherein the aptamer iseither a protein aptamer or a nucleic acid aptamer.
 66. The method ofclaim 64, wherein the aptamer performs a function selected from thegroup consisting of inhibiting expression of a gene, increasingexpression of a gene, inhibiting protein-protein interactions, enhancingprotein-protein interactions, inhibiting the activity of a protein, andenhancing the activity of a protein.
 67. The method of claim 64, whereinthe protein-protein interaction detection system is selected from thegroup consisting of a yeast two-hybrid system, the Ras recruitmentsystem, the split ubiquitin system, and protein fragment complementationsystems.
 68. The method of claim 64, wherein the heterologous gene is ahuman gene.
 69. The method of claim 68, wherein the human gene comprisesa set of alleles, each differing by one or more SNPs.
 70. The method ofclaim 68, wherein the starting yeast strains carry selectable markers topermit efficient recovery of haploid spore progeny.
 71. The method ofclaim 70, wherein the selectable markers are mating type specificpromoters which permit selection of particular haploid mating types. 72.The method of claim 58, wherein robotic manipulation is utilized.